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An epitope tag alters phosphoglycerate dehydrogenase structure and impairs ability to support cell proliferation.

Mattaini KR, Brignole EJ, Kini M, Davidson SM, Fiske BP, Drennan CL, Vander Heiden MG - Cancer Metab (2015)

Bottom Line: Tagged and untagged PHGDH exhibit the same intracellular localization and ability to produce D-2-hydroxyglutarate (D-2HG), an error product of PHGDH, arguing that neither mislocalization nor loss of D-2HG production explains the inability of epitope-tagged PHGDH to support proliferation.Analysis of tagged and untagged PHGDH using size exclusion chromatography and electron microscopy found that an N-terminal epitope tag alters enzyme structure.Purification of untagged recombinant PHGDH eliminates the need to use an epitope tag for enzyme studies.

View Article: PubMed Central - PubMed

Affiliation: Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139 USA ; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 USA.

ABSTRACT

Background: The gene encoding the serine biosynthesis pathway enzyme PHGDH is located in a region of focal genomic copy number gain in human cancers. Cells with PHGDH amplification are dependent on enzyme expression for proliferation. However, dependence on increased PHGDH expression extends beyond production of serine alone, and further studies of PHGDH function are necessary to elucidate its role in cancer cells. These studies will require a physiologically relevant form of the enzyme for experiments using engineered cell lines and recombinant protein.

Results: The addition of an N-terminal epitope tag to PHGDH abolished the ability to support proliferation of PHGDH-amplified cells despite retention of some activity to convert 3-PG to PHP. Introducing an R236E mutation into PHGDH eliminates enzyme activity, and this catalytically inactive enzyme cannot support proliferation of PHGDH-dependent cells, arguing that canonical enzyme activity is required. Tagged and untagged PHGDH exhibit the same intracellular localization and ability to produce D-2-hydroxyglutarate (D-2HG), an error product of PHGDH, arguing that neither mislocalization nor loss of D-2HG production explains the inability of epitope-tagged PHGDH to support proliferation. To enable studies of PHGDH function, we report a method to purify recombinant PHGDH and found that untagged enzyme activity was greater than N-terminally tagged enzyme. Analysis of tagged and untagged PHGDH using size exclusion chromatography and electron microscopy found that an N-terminal epitope tag alters enzyme structure.

Conclusions: Purification of untagged recombinant PHGDH eliminates the need to use an epitope tag for enzyme studies. Furthermore, while tagged PHGDH retains some ability to convert 3PG to PHP, the structural alterations caused by including an epitope tag disrupts the ability of PHGDH to sustain cancer cell proliferation.

No MeSH data available.


Related in: MedlinePlus

PHGDH enzymatic activity is required for cell proliferation following PHGDH knockdown. (A)In vitro enzyme activity assessed by tracking NADH production by fluorescence. The assay was performed at saturating substrate concentrations. (B) Cell number over time for PHGDH-amplified T.T. cells stably expressing shRNA-resistant PHGDH wild type or R236E (enzymatically dead) cDNAs or empty vector (EV) control when infected with virus expressing GFP or PHGDH shRNA. Error bars show standard deviation from the mean.
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Fig2: PHGDH enzymatic activity is required for cell proliferation following PHGDH knockdown. (A)In vitro enzyme activity assessed by tracking NADH production by fluorescence. The assay was performed at saturating substrate concentrations. (B) Cell number over time for PHGDH-amplified T.T. cells stably expressing shRNA-resistant PHGDH wild type or R236E (enzymatically dead) cDNAs or empty vector (EV) control when infected with virus expressing GFP or PHGDH shRNA. Error bars show standard deviation from the mean.

Mentions: Despite an inability to rescue proliferation, recombinant FLAG-His-PHGDH exhibits enzyme activity in vitro (Figure 2A). PHGDH enzyme activity was measured by tracking NADH production when NAD+ and 3-PG were provided at saturating concentrations, with the product PHP removed by coupling the assay to PSAT and providing glutamate (Figure 1A). To confirm that the ability of exogenous PHGDH to rescue endogenous PHGDH knockdown is dependent on enzymatic activity, we generated a mutant that was predicted to be enzymatically inactive. The positively charged arginine residue at position 236 of PHGDH is thought to anchor the carboxyl group of 3-PG. R236 in human PHGDH is analogous to R240 in E. coli PHGDH, which when replaced with an alanine was effective at disrupting bacterial enzyme function [16]. Thus, we determined whether mutation of R236 in human PHGDH to a negatively charged glutamate would disrupt enzyme function. FLAG-His-PHGDH R236E was purified from E. coli and had no enzyme activity in vitro (Figure 2A). We stably overexpressed an shRNA-resistant, untagged version of the R236E mutant in T.T. cells and found that it was incapable of rescuing cell proliferation following knockdown of endogenous PHGDH (Figure 2B and Additional file 1: Figure S1). These data support PHGDH enzymatic function being required for the proliferation of T.T. cells.Figure 2


An epitope tag alters phosphoglycerate dehydrogenase structure and impairs ability to support cell proliferation.

Mattaini KR, Brignole EJ, Kini M, Davidson SM, Fiske BP, Drennan CL, Vander Heiden MG - Cancer Metab (2015)

PHGDH enzymatic activity is required for cell proliferation following PHGDH knockdown. (A)In vitro enzyme activity assessed by tracking NADH production by fluorescence. The assay was performed at saturating substrate concentrations. (B) Cell number over time for PHGDH-amplified T.T. cells stably expressing shRNA-resistant PHGDH wild type or R236E (enzymatically dead) cDNAs or empty vector (EV) control when infected with virus expressing GFP or PHGDH shRNA. Error bars show standard deviation from the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4414297&req=5

Fig2: PHGDH enzymatic activity is required for cell proliferation following PHGDH knockdown. (A)In vitro enzyme activity assessed by tracking NADH production by fluorescence. The assay was performed at saturating substrate concentrations. (B) Cell number over time for PHGDH-amplified T.T. cells stably expressing shRNA-resistant PHGDH wild type or R236E (enzymatically dead) cDNAs or empty vector (EV) control when infected with virus expressing GFP or PHGDH shRNA. Error bars show standard deviation from the mean.
Mentions: Despite an inability to rescue proliferation, recombinant FLAG-His-PHGDH exhibits enzyme activity in vitro (Figure 2A). PHGDH enzyme activity was measured by tracking NADH production when NAD+ and 3-PG were provided at saturating concentrations, with the product PHP removed by coupling the assay to PSAT and providing glutamate (Figure 1A). To confirm that the ability of exogenous PHGDH to rescue endogenous PHGDH knockdown is dependent on enzymatic activity, we generated a mutant that was predicted to be enzymatically inactive. The positively charged arginine residue at position 236 of PHGDH is thought to anchor the carboxyl group of 3-PG. R236 in human PHGDH is analogous to R240 in E. coli PHGDH, which when replaced with an alanine was effective at disrupting bacterial enzyme function [16]. Thus, we determined whether mutation of R236 in human PHGDH to a negatively charged glutamate would disrupt enzyme function. FLAG-His-PHGDH R236E was purified from E. coli and had no enzyme activity in vitro (Figure 2A). We stably overexpressed an shRNA-resistant, untagged version of the R236E mutant in T.T. cells and found that it was incapable of rescuing cell proliferation following knockdown of endogenous PHGDH (Figure 2B and Additional file 1: Figure S1). These data support PHGDH enzymatic function being required for the proliferation of T.T. cells.Figure 2

Bottom Line: Tagged and untagged PHGDH exhibit the same intracellular localization and ability to produce D-2-hydroxyglutarate (D-2HG), an error product of PHGDH, arguing that neither mislocalization nor loss of D-2HG production explains the inability of epitope-tagged PHGDH to support proliferation.Analysis of tagged and untagged PHGDH using size exclusion chromatography and electron microscopy found that an N-terminal epitope tag alters enzyme structure.Purification of untagged recombinant PHGDH eliminates the need to use an epitope tag for enzyme studies.

View Article: PubMed Central - PubMed

Affiliation: Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139 USA ; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 USA.

ABSTRACT

Background: The gene encoding the serine biosynthesis pathway enzyme PHGDH is located in a region of focal genomic copy number gain in human cancers. Cells with PHGDH amplification are dependent on enzyme expression for proliferation. However, dependence on increased PHGDH expression extends beyond production of serine alone, and further studies of PHGDH function are necessary to elucidate its role in cancer cells. These studies will require a physiologically relevant form of the enzyme for experiments using engineered cell lines and recombinant protein.

Results: The addition of an N-terminal epitope tag to PHGDH abolished the ability to support proliferation of PHGDH-amplified cells despite retention of some activity to convert 3-PG to PHP. Introducing an R236E mutation into PHGDH eliminates enzyme activity, and this catalytically inactive enzyme cannot support proliferation of PHGDH-dependent cells, arguing that canonical enzyme activity is required. Tagged and untagged PHGDH exhibit the same intracellular localization and ability to produce D-2-hydroxyglutarate (D-2HG), an error product of PHGDH, arguing that neither mislocalization nor loss of D-2HG production explains the inability of epitope-tagged PHGDH to support proliferation. To enable studies of PHGDH function, we report a method to purify recombinant PHGDH and found that untagged enzyme activity was greater than N-terminally tagged enzyme. Analysis of tagged and untagged PHGDH using size exclusion chromatography and electron microscopy found that an N-terminal epitope tag alters enzyme structure.

Conclusions: Purification of untagged recombinant PHGDH eliminates the need to use an epitope tag for enzyme studies. Furthermore, while tagged PHGDH retains some ability to convert 3PG to PHP, the structural alterations caused by including an epitope tag disrupts the ability of PHGDH to sustain cancer cell proliferation.

No MeSH data available.


Related in: MedlinePlus